Comparative mapping of loci controlling winter survival and related traits in oilseed Brassica rapa and B. napus

Winter survival is an important characteristic of oilseedBrassica that is seeded in the fall in northern climates,and it may be affected by genetic variation for other cold-regulated traits,such as freezing tolerance and vernalization responsive flowering time. Weanalyzed immortalized populations of oilseed Brassica rapa(recombinant inbred lines) and B. napus (double haploidlines) derived from crosses of annual and biennial types in order to comparethe map positions and effects of quantitative trait loci controlling wintersurvival, nonacclimated and acclimated freezing tolerances, and flowering time.The B. napus population was evaluated in multiple winters,and six of the 16 total significant QTL for winter survival were detected inmore than one winter. Correspondence in the map positions of QTL controllingdifferent traits within species provided evidence that some alleles causinggreater acclimated freezing tolerance and later flowering time also contributedto increased winter survival. Correspondence in the map positions of QTLbetween species provided evidence for allelic variation at homologous loci inB. rapa and B. napus. The potentialrole of some candidate genes in regulating these traits is discussed.     

Mapping loci controlling vernalization requirement in Brassica rapa

Brassica cultivars are classified as biennial or annual based on their requirement for a period of cold treatment (vernalization) to induce flowering. Genes controlling the vernalization requirement were identified in a Brassica rapa F₂ population derived from a cross between an annual and a biennial oilseed cultivar by using an RFLP linkage map and quantitative trait locus (QTL) analysis of flowering time in F₃ lines. Two genomic regions were strongly associated with variation for flowering time of unvernalized plants and alleles from the biennial parent in these regions delayed flowering. These QTLs had no significant effect on flowering time after plants were vernalized for 6 weeks, suggesting that they control flowering time through the requirement for vernalization. The two B. rapa linkage groups containing these QTLs had RFLP loci in common with two B. napus linkage groups that were shown previously to contain QTLs for flowering time. An RFLP locus detected by the cold-induced gene COR6.6 cloned from Arabidopsis thaliana mapped very near to one of the B. rapa QTLs for flowering time.     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

Identification of quantitative trait loci and associated candidate genes for low-temperature tolerance in cold-hardy winter wheat

Low-temperature (LT) tolerance is an important economic trait in winter wheat (Triticum aestivum L.) that determines the plants’ ability to cope with below freezing temperatures. Essential elements of the LT tolerance mechanism are associated with the winter growth habit controlled by the vernalization loci (Vrn-1) on the group 5 chromosomes. To identify genomic regions, which in addition to vrn-1 determine the level of LT tolerance in hexaploid wheat, two doubled haploid (DH) mapping populations were produced using parents with winter growth habit (vrn-A1, vrn-B1, and vrn-D1) but showing different LT tolerance levels. A total of 107 DH lines were analyzed by genetic mapping to produce a consensus map of 2,873 cM. The LT tolerance levels for the Norstar (LT₅₀=−20.7°C) × Winter Manitou (LT₅₀=−14.3°C) mapping population ranged from −12.0 to −22.0°C. Single marker analysis and interval mapping of phenotyped lines revealed a major quantitative trait locus (QTL) on chromosome 5A and a weaker QTL on chromosome 1D. The 5A QTL located 46 cM proximal to the vrn-A1 locus explained 40% of the LT tolerance variance. Two C-repeat Binding Factor (CBF) genes expressed during cold acclimation in Norstar were located at the peak of the 5A QTL.     

Quantitative trait analysis of seed yield and other complex traits in hybrid spring rapeseed (Brassica napus L.): 1. Identification of genomic regions from winter germplasm

The introgression of winter germplasm into spring canola (Brassica napus L.) represents a novel approach to improve seed yield of hybrid spring canola. In this study, quantitative trait loci (QTL) for seed yield and other traits were genetically mapped to determine the effects of genomic regions introgressed from winter germplasm into spring canola. Plant materials used comprised of two populations of doubled haploid (DH) lines having winter germplasm introgression from two related French winter cultivars and their testcrosses with a spring line used in commercial hybrids. These populations were evaluated for 2 years at two locations (Wisconsin, USA and Saskatchewan, Canada). Genetic linkage maps based on RFLP loci were constructed for each DH population. Six QTL were detected in the testcross populations for which the winter alleles increased seed yield. One of these QTL explained 11 and 19% of the phenotypic variation in the two Canadian environments. The winter allele for another QTL that increased seed yield was linked in coupling to a QTL allele for high glucosinolate content, suggesting that the transition of rapeseed into canola could have resulted in the loss of favorable seed yield alleles. Most QTL for which the introgressed allele decreased seed yield of hybrids mapped to genomic regions having homoeologous non-reciprocal transpositions. This suggests that allelic configurations created by these rearrangements might make an important contribution to genetic variation for complex traits in oilseed B. napus and could account for a portion of the heterotic effects in hybrids. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Population-based resequencing analysis of improved wheat germplasm at wheat leaf rust resistance locus Lr21

Little is known about the genetic control of heterosis in the complex polyploid crop species oilseed rape (Brassica napus L.). In this study, two large doubled-haploid (DH) mapping populations and two corresponding sets of backcrossed test hybrids (THs) were analysed in controlled greenhouse experiments and extensive field trials for seedling biomass and yield performance traits, respectively. Genetic maps from the two populations, aligned with the help of common simple sequence repeat markers, were used to localise and compare quantitative trait loci (QTL) related to the expression of heterosis for seedling developmental traits, plant height at flowering, thousand seed mass, seeds per silique, siliques per unit area and seed yield. QTL were mapped using data from the respective DH populations, their corresponding TH populations and from mid-parent heterosis (MPH) data, allowing additive and dominance effects along with digenic epistatic interactions to be estimated. A number of genome regions containing numerous heterosis-related QTL involved in different traits and at different developmental stages were identified at corresponding map positions in the two populations. The co-localisation of per se QTL from the DH population datasets with heterosis-related QTL from the MPH data could indicate regulatory loci that may also contribute to fixed heterosis in the highly duplicated B. napus genome. Given the key role of epistatic interactions in the expression of heterosis in oilseed rape, these QTL hotspots might harbour genes involved in regulation of heterosis (including fixed heterosis) for different traits throughout the plant life cycle, including a significant overall influence on heterosis for seed yield.     

Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

Mapping QTL controlling fatty acid composition in a doubled haploid rapeseed population segregating for oil content

Increasing oil content and improving the fatty acid composition in the seed oil are important breeding goals for rapeseed (Brassica napus L.). The objective of the study was to investigate a possible relationship between fatty acid composition and oil content in an oilseed rape doubled haploid (DH) population. The DH population was derived from a cross between the German cultivar Sollux and the Chinese cultivar Gaoyou, both having a high erucic acid and a very high oil content. In total, 282 DH lines were evaluated in replicated field experiments in four environments, two each in Germany and in China. Fatty acid composition of the seed oil was analyzed by gas liquid chromatography and oil content was determined by NIRS. Quantitative trait loci (QTL) for fatty acid contents were mapped and their additive main effects were determined by a mixed model approach using the program QTLMapper. For all fatty acids large and highly significant genetic variations among the genotypes were observed. High heritabilities were determined for oil content and for all fatty acids (h ² = 0.82 to 0.94), except for stearic acid content (h ²= 0.38). Significant correlations were found between the contents of all individual fatty acids and oil content. Closest genetic correlations were found between oil content and the sum of polyunsaturated fatty acids (18:2 + 18:3; r _G = −0.46), the sum of monounsaturated fatty acids (18:1 + 20:1 + 22:1; r _G = 0.46) and palmitic acid (16:0; r _G = −0.34), respectively. Between one and eight QTL for the contents of the different fatty acids were detected. Together, their additive main effects explained between 28% and 65% of the genetic variance for the individual fatty acids. Ten QTL for fatty acid contents mapped within a distance of 0 to 10 cM to QTL for oil content, which were previously identified in this DH population. QTL mapped within this distance to each other are likely to be identical. The results indicate a close interrelationship between fatty acid composition and oil content, which should be considered when breeding for increased oil content or improved oil composition in rapeseed.     

Mapping loci controlling flowering time in Brassica oleracea

The timing of the transition from vegetative to reproductive phase is a major determinant of the morphology and value of Brassica oleracea crops. Quantitative trait loci (QTLs) controlling flowering time in B. oleracea were mapped using restriction fragment length polymorphism (RFLP) loci and flowering data of F₃ families derived from a cabbage by broccoli cross. Plants were grown in the field, and a total of 15 surveys were made throughout the experiment at 5–15 day intervals, in which plants were inspected for the presence of flower buds or open flowers. The flowering traits used for data analysis were the proportion of annual plants (PF) within each F₃ family at the end of the experiment, and a flowering-time index (FT) that combined both qualitative (annual/biennial) and quantitative (days to flowering) information. Two QTLs on different linkage groups were found associated with both PF and FT and one additional QTL was found associated only with FT. When combined in a multi-locus model, all three QTLs explained 54.1% of the phenotypic variation in FT. Epistasis was found between two genomic regions associated with FT. Comparisons of map positions of QTLs in B. oleracea with those in B. napus and B. rapa provided no evidence for conservation of genomic regions associated with flowering time between these species.     

The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves

Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2 degrees C h(-1)) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.     

Behavioural and chemical ecology underlying the success of turnip rape (Brassica rapa) trap crops in protecting oilseed rape (Brassica napus) from the pollen beetle (Meligethes aeneus)

There is increasing interest in the use of trap crops as components of integrated pest management (IPM) strategies. Understanding the mechanisms underlying host plant preferences of herbivorous pests can lead to improved effectiveness and reliability of the trap crop. We investigated the behavioural and chemical ecology underlying the success of turnip rape, Brassica rapa, trap crops in protecting oilseed rape, Brassica napus, from the pollen beetle, Meligethes aeneus, which feeds in the flowers and lays its eggs in the buds causing yield loss. Using a semi-field arena bioassay, plant growth stage was found to be a major factor in the preference of this pest for B. rapa over B. napus. Plants at early-flowering growth stages were preferred over plants in the bud stage, irrespective of species. No preference was found when both species were flowering. As B. rapa develops faster than B. napus in the field, this could explain part of the mechanism of its success as a trap crop. However, B. rapa was preferred over B. napus when both species were in the bud stage, indicating some inherent preferences for B. rapa. Responses of M. aeneus in olfactometer tests to the odours of B. napus and B. rapa at the bud and flowering growth stages, reflected those of the semi-field arena bioassay. These behavioural responses can be explained by volatile compounds associated with the flowering stage. Phenylacetaldehyde, indole and (E,E)-α-farnesene were found to be present in air entrainment samples of both plant species at the flowering growth stage, but only in those of B. rapa at the bud stage. The former two compounds were behaviourally-active in olfactometer tests. These compounds are likely to be involved in host location by M. aeneus, and, at least partially, responsible for the attractiveness of B. rapa and its success as a trap crop to protect B. napus from this pest.     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Identification of two loci for resistance to clubroot (<Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> Woronin) in <Emphasis Type="Italic">Brassica rapa</Emphasis> L.

In an analysis of 114 F₂ individuals from a cross between clubroot-resistant and susceptible lines of Brassica rapa L., 'G004' and 'Hakusai Chukanbohon Nou 7' (A9709), respectively, we identified two loci, Crr1 and Crr2, for clubroot (caused by Plasmodiophora brassicae Woronin) resistance. Each locus segregated independently among the F₂ population, indicating that the loci reside on a different region of chromosomes or on different chromosomes. Genetic analysis showed that each locus had little effect on clubroot resistance by itself, indicating that these two loci are complementary for clubroot resistance. The resistance to clubroot was much stronger when both loci were homozygous for resistant alleles than when they were heterozygous. These results indicate that clubroot resistance in B. rapa is under oligogenic control and at least two loci are necessary for resistance.Communicated by H.C. Becker     

SNP markers‐based map construction and genome‐wide linkage analysis in Brassica napus

An Illumina Infinium array comprising 5306 single nucleotide polymorphism (SNP) markers was used to genotype 175 individuals of a doubled haploid population derived from a cross between Skipton and Ag‐Spectrum, two Australian cultivars of rapeseed (Brassica napus L.). A genetic linkage map based on 613 SNP and 228 non‐SNP (DArT, SSR, SRAP and candidate gene markers) covering 2514.8 cM was constructed and further utilized to identify loci associated with flowering time and resistance to blackleg, a disease caused by the fungus Leptosphaeria maculans. Comparison between genetic map positions of SNP markers and the sequenced Brassica rapa (A) and Brassica oleracea (C) genome scaffolds showed several genomic rearrangements in the B. napus genome. A major locus controlling resistance to L. maculans was identified at both seedling and adult plant stages on chromosome A07. QTL analyses revealed that up to 40.2% of genetic variation for flowering time was accounted for by loci having quantitative effects. Comparative mapping showed Arabidopsis and Brassica flowering genes such as Phytochrome A/D, Flowering Locus C and agamous‐Like MADS box gene AGL1 map within marker intervals associated with flowering time in a DH population from Skipton/Ag‐Spectrum. Genomic regions associated with flowering time and resistance to L. maculans had several SNP markers mapped within 10 cM. Our results suggest that SNP markers will be suitable for various applications such as trait introgression, comparative mapping and high‐resolution mapping of loci in B. napus.     

Genetic mapping and localization of a major QTL for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis)

Downy mildew caused by the fungus Peronospora parisitica is a serious threat to members of the Brassicaceae family. Annually, a substantial loss of yield is caused by the widespread presence of this disease in warm and humid climates. The aim of this study was to localize the genetic factors affecting downy mildew resistance in Chinese cabbage (Brassica rapa ssp. pekinensis). To achieve this goal, we improved a preexisting genetic map of a doubled-haploid population derived from a cross between two diverse Chinese cabbage lines, 91-112 and T12-19, via microspore culture. Microsatellite simple sequence repeat (SSR) markers, isozyme markers, sequence-related amplified polymorphism markers, sequence-characterized amplified region markers and sequence-tagged-site markers were integrated into the previously published map to construct a composite Chinese cabbage map. In this way, the identities of linkage groups corresponding to the Brassica A genome reference map were established. The new map contains 519 markers and covers a total length of 1,070 cM, with an average distance between markers of 2.06 cM. All markers were designated as A1–A10 through alignment and orientation using 55 markers anchored to previously published B. rapa or B. napus reference maps. Of the 89 SSR markers mapped, 15 were newly developed from express sequence tags in Genbank. The phenotypic assay indicated that a single major gene controls seedling resistance to downy mildew, and that a major QTL was detected on linkage group A8 by both interval and MQM mapping methods. The RAPD marker K14-1030 and isozyme marker PGM flanked this major QTL in a region spanning 2.9 cM, and the SSR marker Ol12G04 was linked to this QTL by a distance of 4.36 cM. This study identified a potential chromosomal segment and tightly linked markers for use in marker-assisted selection to improve downy mildew resistance in Chinese cabbage.     

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop × weed hybrid generations

The level of transgene expression in crop × weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T₁ single-locus insert GFP/Bacillus thuringiensis (Bt) transgenic canola (Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC₁F₁, BC₂F₁) were produced by backcrossing various GFP/Bt transgenic canola (B. napus, cv Westar) and birdseed rape (Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC₂F₂ Bulk) were generated by crossing BC₂F₁ individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC₂F₂ Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F₁ hybrid generations contained 95–97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15–29% presence in the BC₂F₂ Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC₂F₂ Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F₁, BC₁F₁ and BC₂F₁). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.Communicated by J. Dvorak